1. Field of the Invention
This invention relates to methods for the determination of the Dibucaine Number (DN) and compositions used in said determination.
2. Description of the Prior Art
Approximately one out of 2,500 persons in a normal population has a genetically determined atypical variant of the enzyme: plasma cholinesterase (PchE, E.C.3.1.1.8.). The enzyme has no known physiological role so this variation is of no consequence in our daily lives. However, the enzyme is responsible for the metabolism of the short-acting muscle relaxant, succinylcholine, a drug used daily by anesthesiologists in over 50% of patients to facilitate intubation of the trachea during the conduct of general anesthesia. In the patient with genetically normal PchE, the duration of complete muscle paralysis from succinylcholine is approximately five to seven minutes; however, in those with atypical enzyme, complete paralysis can persist for several hours. These few patients require mechanical ventilation to sustain life until the drug has been eliminated by non-enzymatic pathways.
The assays for PchE will not distinguish the normal enzyme from the atypical variant. However, the normal can be distinguished from the atypical by assaying PchE activity in the presence and absence of dibucaine (a local anesthetic that also inhibits PchE activity). As shown by Kalow and Genest, a given concentration of dibucaine inhibits the activity of the normal enzyme about 80 percent and the atypical about 20 percent.
The dibucaine number determination, Kalow et al. ("A Method for the Detection of Atypical Forms of Human Serum Cholinesterase. Determination of Dibucaine Numbers.", Can. J. Biochem. Physiol. 35: 339-346 (1957)) has become the classical method for distinguishing genetically normal plasma cholinesterase from an abnormal variant. This determination is of importance to anesthesiologists because it permits prediction of the response of a specific patient to succinylcholine and it also provides needed information when confronted with post-operative apnea. The dibucaine number is calculated from the ratio of activities of the enzyme with and without DIBUCAINE-HCl (C.sub.20 H.sub.30 ClN.sub.3 O.sub.2) 2-butoxy-N-(2-Diethylaminoethyl)cinchoninamide hydrochloride.
Although the original method of Kalow et al is relatively easy to perform, it is time consuming, it requires advance preparation before samples can be assayed, it requires special skills on the part of the technician and, consequently, results do not usually become available until days or weeks after the information is requested. In most cases, the laboratory performing the assay is geographically separated from the requesting physician.
A need therefore, continues to exist for a rapid, simple, semi-automated method for the determination of dibucaine numbers.